Author: Sharma, R K; Gandhi, E
Date published: April 1, 2010
Journal code: JCLT
Lymphocytes are involved in regulation of diverse ovarian functions such as follicle development and growth, follicular atresia, corpus luteumformation and regression . The frequency of lymphocytes varies with ovarian cyclic activity. Leukocyte trafficking operates in and out of ovarian tissue which is influenced by chemokine modulation [2-8]. T lymphocytes secrete an array of cytokines (IL-2, IL- 6, GM-CSF, and IFN's) and are actively involved in remodeling of ovarian tissue during ovulation and corpus luteum regression [9-16].
The distribution of lymphocytes has been analyzed in the ovaries of a number of mammalian species [17-22]. The information generated so far is inconsistent and hence biological significance cannot be ascertained. To the best of the knowledge of authors no information is available on the leukocyte subtypes and their distribution. Keeping in view the existing lacunae in literature, the present investigation was made to establish the interaction of CD4^sup +^ and CD8^sup +^ lymphocytes with ovarian functional attributes.
MATERIALS AND METHODS
Materials: The normal cycling goat ovaries collected from the slaughter houses of Chandigarh (30030'N to 30°45'N, 76°45'E to 76055'E) and were brought to laboratory at 4 °C in normal saline. The ovaries were fixed in Bouins fixative for 24 h at room temperature and processed for paraffin sectioning. The serial 5 µm thick sections were used for immunohistochemistry. Mouse monoclonal antibody (mAb) to T cell subset helper cells CD4 (cat no. AHSO412) and cytotoxic cells CD8 (Cat no. 217580) were used at 1:100 dilutionwith biotinylated goat anti polyvalent IgG as secondary antibody (TP-015BN). A spreptavidin alkaline phosphatase (Labvision corporation - TS-015-AP) method was applied to detect the presence of T-lymphocytes. The specific colour reaction was developed with napthol phosphate (fast red tablets). One slide was stained with haematoxylene and used as control.
Immunohistochemistry: Paraffin embedded sections were deparafinized and rehydrated in alcoholic grades, followed by phosphate buffer saline (PBS) washing. Antigen retrieval was done in citrate buffer (pH 6.0) for 10 minutes in microwave for activating the epitopes. The sections were washed in PBS, before incubation for 5 minutes with ultra V block to block non-specific background staining.After thorough washing in PBS, sections were incubated with mouse mAb to T-cell subset-helper cells CD4 and cytotoxic cells CD8 at a 1:100 dilution in buffer overnight at 4 °C. The sections were incubated with biotinylated secondary antibody (goat antirabbit, IgG) for 30 minutes and steptavidin alkaline phosphate for a further 30 minutes, followed by napthol phosphate substrate (fast red tablets) treatment for 10-20 minutes. Thereafter, the sections were washed with PBS and counterstained with haematoxylene and mounted in glycerol. CD4^sup +^ and CD8^sup +^ cells were evaluated under light microscope.
Variable number of CD4^sup +^ and CD8^sup +^ cells were mapped in the cellular components of the follicles. Both the CD4^sup +^ and CD8^sup +^ cells were observed in primordial, primary, secondary and antral follicles (Table 1). CD4^sup +^ and CD8^sup +^ cells were present in the granulosa and thecal compartments of developing follicles. The primordial follicles were least reactive to both the antibodies. The number of CD4^sup +^ cells was higher as compared to CD8^sup +^ cells in all stages of the developing follicles (Table 1). The granulosa cells were more reactive to CD4^sup +^ than CD8^sup +^, whereas thecal cells were more reactive to CD8^sup +^ than CD4^sup +^. The minimum reactivity for CD4^sup +^ and CD8^sup +^ was observed in primordial follicles whilemax-imum binding was seen in large sized follicles (Table 2). A few of the CD4^sup +^ and CD8^sup +^ cells were also observed in the ovarian stroma surrounding follicles. In atretic follicles, number of both cell types increased from primary to large sized follicles as compared to their counterpart normal developing follicles. A small number of CD4^sup +^ and CD8^sup +^ cells were also found in the blood capillaries around the follicles. CD4^sup +^ cells were also observed in the cumulus oophorus cells.
The frequency of CD4^sup +^ and CD8^sup +^ cells was higher in the atretic follicles at all stages (Table 3). The maximum staining was observed in both normal and atretic dominant follicles, moderate in primary and least or absent in the primordial follicles.
The Immunohistochemical localization of CD4^sup +^ and CD8^sup +^ cells in the goat ovary has revealed their presence in thecal and granulosa layers of the follicles and their frequency increased in the atretic follicles. This is in accordance with the findings of Lei et al. , Wang et al.  and Brannstrom et al.  who demonstrated CD4^sup +^ and CD8^sup +^ cells in human ovaries. The relative frequency of distribution of CD4^sup +^ and CD8^sup +^ cells increased during follicular development and supports the observation of Lutton and Callard  in human ovaries. However, present findings contradict the earlier observations of Brannstrom et al. , who have reported no difference in CD4^sup +^ and CD8^sup +^ cells numbers in follicles throughout the cycle.
Developing follicles contain a small number of CD4^sup +^ and CD8^sup +^ cells in and around the blood vessels of the theca and its number greatly increased in the atretic follicles. This is in accordance with the findings of Pestka et al. , Fukuoka et al. , Wang et al. , Best et al. [20,28], Suzuki et al.  andGayton et al. , who have suggested that these cells may be responsible for macrophage activation and steroid inhibition through the secretion of cytokines. The attraction of leukocytes from the blood vessels by chemo-attractants may involve the local modulation of chemokines expression combined with increased vascular permeability . Present results strongly refute the findings of Droesch et al. , who observed a decrease in the percentage of CD4^sup +^ and CD8^sup +^ cells with increase in follicles size.
The present study has demonstrated higher frequency of CD4^sup +^ and CD8^sup +^ cells both in the granulosa and thecal layers of atretic follicles as compared to normal follicles of goat ovary. This increased number of CD4^sup +^ and CD8^sup +^ sites can be attributed to infiltration of leukocytes which were frequently observed, suggesting the involvement of leukocytes in the process of clearing of dead cells during the course of follicular atresia in normal cycling goat ovaries.
The authors acknowledge University Grants Commission, NewDelhi for financial support and also thankful to Kurukshetra University, Kurukshetra for the laboratory facilities.
 Brannstrom,M. and Norman, R.J.: Hum. Reprod., 8: 1762-1775(1993).
 Espey, L.L.: Biol. Reprod., 22: 73-106 (1980).
 Espey, L.L.: Biol.Reprod., 50: 233-238 (1994).
 Machelon, V. and Emilie, D.: Eur. CytokineNetwork, 8: 137-143 (1997).
 Simon, C., Caballero-Campo, P., Garcia-Velasco, J.A. andPellicer,A.: J.Reprod.Immunol., 38: 169-193 (1998).
 Bukulmez, O. and Arici, A.: Hum. Reprod., 6: 1-15 (2000).
 Brannstrom,M. and Enskog,A.: J. Reprod. Immunol., 57:47-60 (2002).
 Wu, R., Vander Hoek, K.H., Ryan, N.K., Noman, R.J. and Roker,R.L.: Hum.Reprod., 10(2): 119-133 (2004).
 Carlberg,M.,Nejaty, J., Froysa, B., Guan,Y., Soder, O. andBergqvist,A.:Hum.Reprot., 15: 1250-1255 (2000).
 Kawano, Y., Kawasaki, F., Nakamura, S., Matsui, N., Narahara, H. and Miyakawa, I.: Am. J. Reprod. Immunol., 45: 1-5 (2001).
 Robert, C.W.,Walker,W. andAlenxander, J.: Clinical. Microbiol.Rev., 14: 3476-3488 (2001).
 Fujii,A.,Harada, T.,Yamanchi,N., Iwabe, T.,Nishi,Y., Yanase,T.,Nawata, H. andTerekawa,N. : Fertil. Steril., 79:151-157 (2003).
 Tanriverdi, F., Silveira, L.F.,MacColl,G.S. andBouloux, P.M.: J. Endocrinol., 176: 3293-3304 (2003).
 Townson, D.H. and Liptak, A.R.: Reprod. Biol. Endocrinol., 1: 94 -100 (2003).
 Itoh, M., Terayama, H., Naito, M., Ogawa, Y. and Tainosho., S.: J. Reprod. Immunol., 67: 1257-1267 (2005).
 Wira, C.R., Fashey, J.V., Sentaman, C.L., Pioli, P.A. and Shen, L.: Immunol.Rev., 206: 306-335 (2005).
 Petrovska, M., Sedlak, R. and Nouza, K.: Am. J. Reprod. Immunol., 28: 77-80 (1992).
 Wang, L.J., Pascoe, V. and Petrucco, O.M.: Hum. Reprod., 7: 197-202 (1992).
 Bukovsky, A., Caudle,M.R. and Keenan, J.A.: Biol. Reprod., 53: 1373-1384 (1995).
 Best, C.L., Pudney, J.,Welch,W.R.:Hum. Reprod., 11: 790-797(1996).
 Miracle,A.L.,Anderson,M.K., Litman, R.T.,Walsh, C.J., Luer, C.A., Rothenberg, E.V., Litman, G.W.: Int. Immunol., 13: 4567-4580 (2001).
 Rumfelt, L.L.,Mckinney, E.C., Taylor, E. and Flajnik, M.F.: Scand. J. Immunol., 56: 2130-2148 (2002).
 Lei, Z.M., Chegini, N. and Roa, CH.V.: Biol. Reprod., 44: 1148-1156 (1991).
 Brannstrom,M., Pascoe,V.,Norman, R.J. andMcclure, N.:Fertil. Steril., 61: 488-495 (1994),.
 Lutton, B. and Callard, I.: Integrative. Comp. Biol., 46(6): 1060-1071 (2006).
 Pestka, S., Langer, J.A., Zoon,K.C. and Samuel, C.E.: Annu.Rev.Biochem., 56: 727-777 (1987).
 Fukuoka, M., Yasuda, K. and Emi, N.: J. Clin. Endocrinol.Metab., 75: 254-258 (1992).
 Best, C.L., Griffin, P.M. andHill, J.A.:Am. J. Obstet. Gynecol., 172: 1505-1510(1995).
 Suzuki, T., Sasano, H., Takaya, R., Fukay, T.,Yajima, A., Date, F. and Nagura, H.: Hum. Reprod., 8: 2186- 2191 (1998).
 Gaytan, F., Morales, C., Bellido, C., Aguilar, E. and Sanchez-Criado, J.E.:Biol.Reprod., 58: 152-159 (1998).
 Machelon, V., Nome, F. and Emilie, D.: J. Clin. Endocrinol.Metab. 85: 1417-1424 (2000).
 Droesch, K., Fulgham,D.L., Liu, H.C., Rosenwaks, Z. andAlexander, N.J.: Fertil. Steril., 50: 618-621 (1988).
SHARMA, R. K.* AND GANDHI, E.
Department of Zoology, Kurukshetra University, Kurukshetra -136119.
E-mail : firstname.lastname@example.org
Received: February 22, 2010; Accepted:March 25, 2010